The high fidelity of double-strand DMA break repair by homology-dependent recombination arises from the use of a second, homologous strand of DMA as a template to repair the broken strand. Illegitimate recombination occurs when the recombination machinery repairs the DNA break using an incorrect template, which will almost certainly result in mutations including translocations and deletions. Genetic screens to identify suppressors of illegitimate recombination in S. cerevisiae revealed roles for the mismatch recognition enzymes, a RecQ family helicase, and endo- and exonucleases. In humans, mutations in the mismatch recognition proteins and several of the RecQ family helicases results in a high incidence of cancer;the role of these proteins in the fidelity enforcement of homologous recombination may account for some of the increased cancer susceptibility of the carriers of these mutations. While these studies have lead to the proposal of several models, these genetic studies cannot determine when and where in the recombination pathway these proteins act, or what specific mechanism(s) they employ to prevent the progression of illegitimate recombination intermediates. I will investigate this process biochemically, using purified human proteins and DNA substrates mimicking recombination intermediates. With these reagents, I will use traditional ensemble methods as well as single molecule approaches to determine when and how the mismatch recognition complexes identify potential illegitimate recombination intermediates, as well as investigate how the binding of these complexes influences the fate of the bound intermediates. These studies will shed light upon an important, yet poorly understood aspect of DNA recombination. There is a well established link between mutations in the genes involved in homologous recombination and cancer. The results from the proposed experiments will provide revolutionary new information about how the components of homologous recombination cooperate to ensure the high fidelity of this DNA repair process. Understanding the function of these proteins, and how mutations in these genes predispose affected individuals to cancer, has the potential to provide new therapeutic strategies for these patients.